This continuation proposal is aimed at the design, preparation, and testing of monolithic "molded" separation media in entirely new shapes and formats that complement current bead-based packed media and provide new and unmatched capabilities for the study, detection, isolation, and separation of biologically active molecules. The new separation media are based on a continuous body of macroporous polymer molded directly into the housing of the separation device rather than made of small beads mechanically packed into the column. The "molded" media offer many advantages such as ease of preparation and ease of handling, reproducibility, versatility of separation chemistry, unmatched ability to incorporate gradients of porosity and of chemistry, compactness, and simple scale-up. In contrast to the two-phase systems characteristic of the suspension polymerization used to prepare bead-shaped separation media, the molded media are prepared from a single phase allowing the use of many functional or water-soluble monomers that cannot be used for conventional beads. The ease of preparation is another important factor that removes the size fractionation and packing steps which are known to greatly affect the efficiency of packed columns. The preparation of the continuous media is simple, waste-free, and less labor-intensive that the preparation of beads, leading to a low unit cost per device. The versatility of separation chemistry is further enhanced by the ability to blend several chemistries or achieve gradients of composition and/or porosity in a single molded medium. Other advantages of the new molded monoliths include remarkably enhanced mass transfer because all of the liquid phase flows through their large pores that are connected to a network of small pores with a large surface area. This allows extremely fast separations of biopolymers and leads to a dramatic improvement in kinetics for immobilized biocatalysts. Overall, our targets are to significantly enhance the array of analytical, preparative, and diagnostics tools available for handling biological fluids. The new media we develop will contribute to both better performance and new capabilities in areas such as high-performance liquid chromatography, electrochromatography, capillary and free-flow zone electrophoresis, diagnostics, detection, enzyme immobilization, etc.. The unequaled versatility of the molded systems and their unique features make them ideal for a broad range of products from the smallest capillary format to large preparative systems for the separation of drugs, peptides, proteins, and oligo- or polynucleotides.